Abstract
The physical mapping of functional genes and polymorphic markers is obviously important for understanding genome organization in higher organisms. Fluorescence in situ hybridization of labeled DNA to metaphase chromosome spreads has been a very effective means of accomplishing this goal (1,2), but it is difficult to routinely detect sequences <3–5 kb in length using this technique (3). Since the polymerase chain reaction (PCR) (4) provides a means to amplify a single copy of DNA to literally hundreds of thousands of copies in a short time, in principle it could be utilized to detect very short sequences directly on metaphase chromosomes. It has been demonstrated that PCR can be performed on the surface of a slide containing cells (5) and that the PCR process can incorporate biotin-dUTP into the amplification product (6). On-slide PCR with a single primer has been used to detect low-copy sequences on metaphase chromosomes (3). Oligonucleotide premed in situ synthesis (PRINS) (7), in which an oligonucleotide is annealed to denatured chromosomes and extended by a DNA polymerase, has also proven to be an effective method to detect low-copy sequences.
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References
Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D. C. (1988) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224–234.
Lichter, P., Tang, C. C., Call, K., Hermanson, G., Evans, G. A., Housman, D., and Ward, D. C. (1990) High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64–69.
Gosden, J. and Hanratty, D. (1993) PCR in situ: a rapid alternative to in situ hybridization for mapping short, low copy number sequences without isotopes. BioTechniques 15(1), 78–80.
Mullis, K. B., Faloona, F., Scharf, S., Saiki, R. K., Horn, G. T., and Erlich, H. A. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263.
Yap, E. P. H. and McGee, J. O. D. (1991) Slide PCR: DNA amplification from cell samples on microscopic glass slides. Nucleic Acids Res. 19, 4294.
Lo, Y. M., Mehal, W. Z., and Fleming, K. A. (1990) Incorporation of biotinylated dUTP, in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic, San Diego, pp. 113–118.
Koch, J., Hindkjaer, J., Mogensen, J., Kölvraa, S., and Bolund, L. (1991) An improved method for chromosome-specific labeling of alpha satellite DNA in situ using denatured double stranded DNA probes as primers in a PRimed IN Situ (PRINS) procedure. Genet. Anal. Techn. Appl. 8, 171–178.
Troyer, D. L., Xie, H., Goad, D. W., and Skinner, D. Z. (1994) Use of a new technique to map the porcine I interferon gene to chromosome 1. Mammalian Genome 5, 112–114.
Troyer, D. L., Goad, D. W., Xie, H., Rohrer, G. A., Alexander, L. J., and Beattie, C. W. (1994) Use of direct in situ single copy (DISC) PCR to physically map 5 porcine microsatellites. Cytogenet. Cell Genetics 67(3), 199–204.
Troyer, D. L., Xie, H., and Goad, D. W. (1995) Use of DISC-PCR to map a porcine microsatellite. Anim. Biotechnol. 6(1), 51–58.
Xie, H., Alexander, L. J., Rohrer, G. A., Beattie, C. W., and Troyer, D. L. (1995) Use of DISC-PCR and FISH to assign a linkage group to pig chromosome 10. Mammalian Genome 6, 139–141.
Litt, M. and Luty, J. A. (1989) A hypervariable microsatellite revealed by in-vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44, 397–401.
Weber, J. L. and May, P. E. (1989) Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44, 338–396.
Schmutz, S. M., Cornwell, D., Moker, J. S., and Troyer, D. L. (1996) Physical mapping of SOD1 to bovine chromosome 1. Cytogenet. Cell. Genetics 72, 37–39.
Halnan, C. R. E. (1989) Banding methods, in Cytogenetics of Animals (Halnan, C. R. E., ed.), CAB International, Wallingford, UK, pp. 451–456.
Ewens, W. J., Griffiths, R. C., Ethier, S. N., Wilcox, S. A., and Marshall Graves, J. A. (1992) Statistical analysis of in situ hybridization data: derivation and use of the Zmax test. Genomics 12, 675–682.
Lemieux, N., Dutrillaux, B., and Viegas-Pequignot, E. (1992) A simple method for simultaneous R-or G-banding and fluorescence in situ hybridization of small single-copy genes. Cytogenet. Cell Genetics 59, 311–312.
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© 1997 Humana Press Inc, Totowa, NJ
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Troyer, D.L., Xie, H., Hu, J. (1997). Direct In Situ Single-Copy (DISC)-PCR. In: Gosden, J.R. (eds) PRINS and In Situ PCR Protocols. Methods in Molecular Biology™, vol 71. Humana Press. https://doi.org/10.1385/0-89603-395-3:71
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DOI: https://doi.org/10.1385/0-89603-395-3:71
Publisher Name: Humana Press
Print ISBN: 978-0-89603-395-5
Online ISBN: 978-1-59259-557-0
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